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Recombinant Hemagglutinin (rHA)
Influenza viruses, commonly known as the flu, belong to the Orthomyxoviridae family of viruses. Hemagglutinin (HA) is a glycoprotein found on the surface of influenza viruses. It is composed of two polypeptides—HA1 and HA2—that are the target of neutralizing antibodies. Hemagglutinin serves two purposes: to bind to sialic acid receptors and to facilitate in membrane fusion (1). Sialic acid is found on the outside of human upper respiratory tracts cells and red blood cells; hemagglutinin is able to bind to these cells. Once bound, hemagglutinin allows the viral genome to enter the host cell by fusing the host endosomal membrane with the viral membrane. There are at least 18 types of hemagglutinin, designated types H1 through H18; only types H1, H2, and H3 are found in influenza viruses that infect humans (1). Recombinant Hemagglutinin (rHA) Recombinant hemagglutinin (rHA) is hemagglutinin that has been produced through recombinant protein DNA technologies. This means the hemagglutinin has been produced through recombinant genetic techniques in a controlled, laboratory setting, resulting in gene sequences that are not found in the natural world. The rHA used specifically for vaccine-development may contain genetic modifications itself, or be marked with certain ‘tags,’ to enhance protein stability (2). In laboratory settings, yeast cells, bacterial cells, insect cells, plant cells, and human cell lines have been used to produce rHA. Several protein expression systems can be used to produce rHA, but regardless of the method, one major criterion must be met: the rHA produced in a lab must resemble the viral hemagglutinin produced during an active infection to yield good results (1; 2). Baculovirus Expression Vector System (BEVS) What are Baculoviruses Baculoviruses are a family of viruses that exclusively infect insects; mammalian and plant cells cannot be infected with the virus. Baculoviruses are composed of double-stranded DNA that is circular and supercoiled, and is contained in a rod-shaped capsid (5). Since the 1990s, these viruses have been used in specific protein expression systems to produce eukaryotic proteins, mainly recombinant glycoproteins (6). What is the BEVS The Baculovirus Expression Vector System (BEVS) is a recombinant protein expression system that allows viruses modified with a gene of interest to produce specific proteins. The most common insect virus used in foreign gene expression is the Autographa californica ''multiple nuclear polyhedrosis virus (AcMNPV)'' (5). ''During viral engineering, the gene of interest is interested into the genome of the baculovirus; the polyhedron gene, which is naturally occurring in the virus, is replaced with the recombinant gene of desired interest. During the last phase of viral infection, the inserted genes fall under the transcriptional control of the AcMNPV polyhedron promoter. Thus, the recombinant genes are being regulated and transcribed by the natural promoter, and synthesis of the recombinant protein occurs (3). These virally-infected cells are incubated in a fermenter for 48 to 72 hours. Once the cells are at least 90% viable and enough quantity of protein has been produced, the cells are harvested. After harvesting, purification, and sterile filtering, the protein can be formulated with PBS and produced into a vaccine (or sold for other commercial purposes) (3; 5). There are several advantages to using the BEVS. Both small-scale and large-scale production of proteins can be performed at low costs. Recombinant proteins can easily be separated from the viruses and recovered from insect cells. Lastly, since the baculoviruses can only infect insect cells, there is no risk associated with manipulating the virus (5). Insect Cell Lines The cell line cultured to produce rHA is the Sf9 insect cell line. This cell line originated from the ovarian tissue of the fall army worm, ''Spodoptera frugiperda. S9f cells are regular in size and have a slightly granular appearance; they double in a 20 to 24 hour period. S9f cells can be grown in serum-containing media or serum-free media; 28 degrees Celsius is the optimal growth temperature, and they do not require any CO2 (7). Purification of rHA rHA is a secreted viral protein. The cell culture is centrifuged down to create a compacted pellet, which is disposed, and remaining supernatant. To remove the virus, the supernatant is filtered through membrane filtration or anion exchange. To concentrate the protein, the filtered material may undergo dialysis, more membrane filtration, or be filtered through affinity chromatography columns (3; 5). FluBlok FluBlock is an FDA-approved vaccine against the influenza viruses. The vaccine is manufactured using rDNA technologies and the BEVS; it contains three full-length rHA proteins that protect against two Type A influenza strains and one Type B strain. FluBlok is approved to prevent the seasonal flu in people ages 18 to 49. FluBlock is manufactured by Protein Sciences Corporation of Meriden, Connecticut (4). References 1) Hemagglutinin (influenza). Wikipedia. http://en.wikipedia.org/wiki/Hemagglutinin_(influenza). Updated May 5, 2014. Accessed September 21, 2014. 2) Santiago FW, Emo KL, Fitzgerald T, et. al. Antigenic and immunogenic properties of recombinant hemagglutinin proteins from H1N1 A/Brisbane/59/07 and B/Florida/04/06 when produced in various protein expression systems. ''Vaccine. ''June 29, 2012; 30(31): 4606-4616. doi: 10.1016/j.vaccine.2012.05.005 3) Cox MJM. Recombinant protein vaccines produced in insect cells. World Health Organization. http://www.who.int/immunization/research/meetings_workshops/Manon_Cox.pdfPublished January 25, 2013. Accessed September 21, 2014. 4) FDA approves new seasonal influenza vaccine made using novel technology. Federal Drug Administration. http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm335891.htm. Published January 16, 2013. Accessed September 21, 2014. 5) Invitrogen by Life Technologies. Guide to Baculovirus Expression Vector Systems (BEVS) and Insect Cell Culture Techniques. http://tools.lifetechnologies.com/content/sfs/manuals/bevtest.pdf. Published February 27, 2002. Accessed September 21, 2014. 6) Baculovirus. Wikipedia. http://en.wikipedia.org/wiki/Baculovirus. Updated Marsh 23, 2014. Accessed September 21, 2014. 7) Invitrogen by Life Technologies. Growth and Maintenance of Insect Cell Lines. http://tools.lifetechnologies.com/content/sfs/manuals/insect_man.pdf. Published January 23, 2013. Accessed September 21, 2014.